Pyrimidine biosynthesis is regulated in a cell-cycle specific manner to ensure sufficient pools for DNA synthesis. At the core of this regulation is the gene CAD in mammals and its homologue, rudimentary (r) in Drosophila melanogaster. These genes encode the first three enzymes in the pyrimidine biosynthetic pathway, including the rate-limiting step, carbamyiphosphate synthetase. The enhancer of rudimentary gene, e(r), was isolated as a regulatory gene of r. It encodes a highly conserved protein, whose function within the cell has not been determined. The maternal and embryonic expression patterns of e(r) suggest an embryonic function in the cell cycle. Other clues to the function and regulation of the e(r) protein (ER) are the presence of three casein kinase II sites on the protein and the binding of the protein to the ribosomal protein S3 (RPS3). The long-term goals of this project are to determine the function of e(r) in growth and development and to elucidate its mode of action in the cell. In this project, the role of the conserved casein kinase II phosphorylation sites in the regulation of the activity of ER will be determined by mutating the sites to either glutamic acid to mimic phosphorylation or to alanine to mimic the unphosphorylated residue. Each mutant gene will then be assayed for activity by its ability to rescue the mutation e(r)pl. Second, the regions of ER and RPS3 that interact will be determined by testing the ability of fragments of each protein to interact in a yeast two-hybrid assay. In this assay, interacting clones will be seen as LEU2 LACZ+ colonies. Finally, null mutations of e(r) will be isolated using a gene-targeting procedure that will utilize a mutant episomal copy of e(r) to recombine with the resident allele. The expression data of e(r) suggest that mutants may be embryonic lethals. If this is the case, the mutants will be analyzed for cellular defects in the embryo that that will help uncover the normal function of e(r).